Economically viable ethanol production from the hemicellulose fraction of plant biomass requires the simultaneous conversion of both pentoses and hexoses at comparable rates and with high yields. Yeasts, in particular Saccharomyces spp., are the most appropriate candidates for this process since they can grow fast on hexoses, both aerobically and anaerobically. Furthermore they are much more resistant to the toxic environment of lignocellulose hydrolysates than (genetically modified) bacteria.
In previous studies evidence has been provided that metabolic engineering of S. cerevisiae for xylose utilization, should be based on the introduction of xylose isomerase (XI, EC 5.3.1.5) (Bruinenberg et al., 1983, Eur J. Appl. Microbiol. Biotechnol. 18:287-292). In contrast to strains that are based on xylose reductase (XR, EC 1.1.1.21) and xylitol dehydrogenase (XD, EC 1.1.1.9), strains expressing XI activity display high alcohol yields and hardly produce xylitol as has recently been demonstrated in WO 03/0624430 and Kuyper et al. 2004, FEMS Yeast Res. 4:655-664. From a theoretical point of view this is not surprising since the route via XR and XD leads to an obstruction in the NADH balance that in the absence of oxygen, can be relieved e.g., via xylitol formation.
WO03/0624430 discloses that the introduction of a functional Piromyces XI into S. cerevisiae allows slow metabolism of xylose via the endogenous xylulokinase (EC 2.7.1.17) encoded by XKS1 and the enzymes of the non-oxidative part of the pentose phosphate pathway and confers to the yeast transformants the ability to grow on xylose.
Kuyper et al. (supra) describe S. cerevisiae strains in which the Piromyces XI has been introduced and which are thereafter subjected to directed evolution in shake flasks show improved rates of xylose fermentation, but still required oxygen for growth. Further selection via a regime of extreme oxygen limitation under xylose excess, followed by anaerobic selection resulted in a laboratory strain (RWB202-AFX) which fulfils at least one of the prerequisites for hemicellulose utilization, namely an acceptable ethanol yield on xylose. However, the specific rate of ethanol production in this strain is still unacceptably low. In particular, the specific sugar consumption rate during growth on xylose (345 mg xylose/g biomass/h) is still ten-fold lower than on glucose. Attempts to further improve strain RWB202-AFX via evolutionary engineering have failed so far.
WO03/0624430 lists a number of alternative genetic modifications that may result in further improvement of the specific rates of ethanol production and/or sugar consumption on xylose in host cells expressing the Piromyces XI gene to a level that would be required for commercial hemicellulose utilization. These alternatives include: (a) increase transport of xylose into the host cell; (b) increased xylulose kinase activity; (c) increased flux of the pentose phosphate pathway; (d) decreased sensitivity to catabolite repression; (e) increased tolerance to ethanol, osmolarity or organic acids; and, (f) reduced production of by-products (such as e.g., xylitol, glycerol and/or acetic acid). More specifically, WO03/0624430 suggests to overexpress one or more of the genes encoding a hexose or pentose transporter, a xylulose kinase (such as the S. cerevisiae XKS1) an enzyme from the pentose phosphate pathway such as a transaldolase (TAL1) or a transketolase (TKL1) glycolytic enzymes, ethanologenic enzymes such as alcohol dehydrogenases, and/or to inactivate a hexose kinase gene, e.g., the S. cerevisiae HXK2 gene, the S. cerevisiae MIG1 or MIG2 genes, the (nonspecific) aldose reductase genes such as the S. cerevisiae GRE3 gene, or genes for enzymes involved in glycerol metabolism such as the S. cerevisiae glycerol-phosphate dehydrogenase 1 and/or 2 genes. WO03/0624430 however does not disclose which of these many alternatives actually does produce an improvement in the specific rates of ethanol production and/or xylose consumption in host cells carrying the Piromyces XI gene.
Karhumaa et al. (2004, “Development of a Xylose-growing Saccharomyces cerevisiae strain expressing bacterial xylose isomerase”, Poster presentation at 2nd Meeting on Physiology of Yeasts and Filamentous Fungi; Mar. 24-28, 2004 Anglet, France, p 43; and, “New Xylose-growing Saccharomyces cerevisiae strain for biofuel ethanol production”, Oral presentation at the 26th Symposium on Biotechnology for Fuels and Chemicals, May 9-12, 2004 Chattanooga Tenn., USA, p. 19) disclose a strain of S. cerevisiae expressing a bacterial XI from Thermus thermophilus. The strain further contains a number of the genetic modifications suggested in WO03/0624430: overexpression of xylulose kinase and all four enzymes of the non-oxidative pentose phosphate pathway as well as inactivation of the S. cerevisiae nonspecific aldose reductase gene (GRE3). However, despite these genetic modifications this strain is incapable of growth on xylose. Only after adaptation to aerobic growth on xylose a strain, TMB3050, was obtained that is capable of growth on xylose at a low rate (μ=0.04 h−1) and with a low specific xylose consumption rate of 4.3 mg xylose/g cells/h. Since undefined genetic modifications (accumulated during adaptation) are clearly required for growth on xylose in the first place, one cannot deduce from the work of Karhumaa et al. (supra) which, if any, of the defined genetic modifications (such as overexpression of xylulose kinase or any of the pentose phosphate pathway enzymes or inactivation of the aldose reductase gene) actually contribute to the ability of the adapted strain to grow on xylose.
It is therefore an object of the present invention to provide for eukaryotic host cells, such as fungal host cells, that are transformed with a XI gene that confers the ability to grow on xylose and which host cells have specific rates of xylose consumption and/or product (ethanol) formation that are compatible with commercial application of the host cells.